| On the basis of establishing the simple and rapid method of separating and identifying C. perfringens, the epidemiology of C. perfringens were investigated in E. davidianus, the isolates of C. perfringens were further identified by the nested PCR, and the genotype and the production of the intestinal toxin were identified by detection of the toxin excreting from the C. perfringens isolates using PCR. Those will be important for the prevention and treatment of C. perfringens disease in E. davidianus.Experiment1Establishment and primary application of isolating and identifying C. perfringens in the feces of E. davidianusTo establish a simple and fast method of isolating and identifying C. perfringens in the feces of E. davidianus, the colony and cultural characteristics of C. perfringens were explored on the nutrient agar medium, mannitol egg yolk agar medium, and blood agar medium. The infection of C. perfringens in E. davidianus in Beijing Nanhaizi E. davidianus Park and Jiangsu Dafeng E. davidianus National Nature Reserve was investigated after three methods of isolating and identifying C. perfringens were compared. And the bio-chemical reaction was used to identify the strains. The results showed that C. perfringens, which was white translucent mycelium with unregular periphery in the nutrient agar medium, was inculated in the LB medium. After that, C. perfringens were identified Gram-positive and inculated on the mannitol egg yolk agar medium and blood agar medium, which showed the pink volcanic crater mycelium with opal turbidity maximum of lecithase. Using the method, the positive rate of C. perfringens in the feces of E. davidianus was26.45%(41/155), including13.04%(3/23) in Beijing Nanhaizi E. davidianus Park and28.79%(38/132) in Jiangsu Dafeng E. davidianus National Nature Reserve. The biochemistry results showed that the eleven strains were C. perfringens. In conclusion, the method used to investigate the infection of C. perfringens in the feces of E. davidianus was simple, fast, accurate and reliability, and the prevelance of C. perfringens in E. davidianus was so high that we must keep our eyes on the epidemiology, prevention and treatment of C. perfringens in E. davidianus.Experiment2Identification of C. perfringens isolated from E. davidianus by nested PCRThree primers were designed acording to the16S rRNA of C. perfringens in GenBank. The nested PCR used to detect the16S rRNA of C. perfringens was established by identifying the primers, detecting the sensitivity and the sensitivity, and used to identify the C. perfringens isolates from E. davidianus. The results showed that the primers were highly differential to C. perfringens, the least dosages detected by nested PCR were ten cfu/mL, and a total of41(100%) samples were all detected positive. In conclusion, nested PCR assay based on16S rRNA, which was established for detection of C. perfringens, can be used as a rapid, simple and convenient tool for the quantitative detection of C. perfringens, with great sensitivity and differentia.Experiment3Identification of C. perfringens genotype and the bacterium excreting enterotoxinFive sets of primers were designed based on the sequences of C. Perfringens toxin genes (cpb, etx, iA, cpb2and cpe) published in GeneBank to genotype all identified isolates and the bacterium excreting C. perfringens enterotoxin by PCR. The results showed that there was no isolate excreting cpb and cpb2, five isolates excreting etx toxin and fifteen isolates excreting iA toxin were found. According to the relationship between the CP genotypes and the toxin types, there were5(12.20%) C. perfringens type D,15(36.59%) isolates type E and21(51.22%) isolates type A. And there were15(36.59%) isolates excreting C. perfringens enterotoxin, including8type A (19.51%) and7type E (17.07%). In conclusion, the primary genotype infecting E. davidianus were C. perfringens type A and E, and only few isoltes could excrete C. perfringens enterotoxin. |
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